Efficient scar-free knock-ins of several kilobases in plants by engineered CRISPR-Cas endonucleases  

作　　者：Tom Schreiber Anja Prange Petra Schafer Thomas Iwen Ramona Grutzner Sylvestre Marillonnet Aurelie Lepage Marie Javelle Wyatt Paul Alain Tissier 

机构地区：[1]Department of Cell and Metabolic Biology,Leibniz Institute of Plant Biochemistry,Weinberg 3,06120 Halle(Saale),Germany [2]Limagrain,Centre de Recherche,Route d’Ennezat,CS 90126,63720 Chappes,France

出　　处：《Molecular Plant》2024年第5期824-837,共14页分子植物（英文版）

基　　金：funded by grant no.031B0548 in the frame of the program"Crop plants of the future"from the Bundesministerium fur Bildung und Forschung to A.T.;funded by the Investissement d’Avenir program of the French National Agency of Research for the project GENIUS(ANR-11-BTBR-0001_GENIUS).

摘　　要：In plants and mammals,non-homologous end-joining is the dominant pathway to repair DNA doublestrand breaks,making it challenging to generate knock-in events.In this study,we identified two groups of exonucleases from the herpes virus and the bacteriophage T7 families that conferred an up to 38-fold increase in homology-directed repair frequencies when fused to Cas9/Cas12a in a tobacco mosaic virus-based transient assay in Nicotiana benthamiana.We achieved precise and scar-free insertion of several kilobases of DNA both in transient and stable transformation systems.In Arabidopsis thaliana,fusion of Cas9 to a herpes virus family exonuclease led to 10-fold higher frequencies of knock-ins in the first generation of transformants.In addition,we demonstrated stable and heritable knock-ins in wheat in 1%of the primary transformants.Taken together,our results open perspectives for the routine production of heritable knock-in and gene replacement events in plants.

关 键 词：homology-directed repair knockin gene replacement CRISPR-Cas 50 exonuclease PLANTS 

分 类 号：Q94[生物学—植物学]